Citation: Wang, Y.; Ral, J.-P.; Saulnier, L.; Kansou, K. How Does Starch Structure Impact Amylolysis? Review of Current Strategies for Starch Digestibility Study. Foods 2022, 11, 1223. https://doi.org/10.3390/ foods11091223 Academic Editors: Isabel Hernando and Amparo Quiles Received: 4 April 2022 Accepted: 21 April 2022 Published: 24 April 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). foods Systematic Review How Does Starch Structure Impact Amylolysis? Review of Current Strategies for Starch Digestibility Study Yuzi Wang 1 , Jean-Philippe Ral 2 , Luc Saulnier 1 and Kamal Kansou 1, * 1 INRAE, UR1268, Biopolymers, Interactions & Assemblies (BIA), 44316 Nantes, France; yuzi.wang@inrae.fr (Y.W.); luc.saulnier@inrae.fr (L.S.) 2 CSIRO Agriculture and Food, GPO Box 1700, Canberra, ACT 2601, Australia; jean.ral@csiro.au * Correspondence: kamal.kansou@inrae.fr; Tel.: +33-02-40-67-51-49 Abstract: In vitro digestibility of starch is a common analysis in human nutrition research, and generally consists of performing the hydrolysis of starch by α-amylase in specific conditions. Similar in vitro assays are also used in other research fields, where different methods can be used. Overall, the in vitro hydrolysis of native starch is a bridge between all of these methods. In this literature review, we examine the use of amylolysis assays in recent publications investigating the complex starch structure-amylolysis relation. This review is divided in two parts: (1) a brief review of the factors influencing the hydrolysis of starch and (2) a systematic review of the experimental designs and methods used in publications for the period 2016–2020. The latter reports on starch materials, factors investigated, characterization of the starch hydrolysis kinetics and data analysis techniques. This review shows that the dominant research strategy favors the comparison between a few starch samples most frequently described through crystallinity, granule type, amylose and chain length distribution with marked characteristics. This strategy aims at circumventing the multifactorial aspect of the starch digestion mechanism by focusing on specific features. An alternative strategy relies on computational approaches such as multivariate statistical analysis and machine learning techniques to decipher the role of each factor on amylolysis. While promising to address complexity, the limited use of a computational approach can be explained by the small size of the experimental datasets in most publications. This review shows that key steps towards the production of larger datasets are already available, in particular the generalization of rapid hydrolysis assays and the development of quantification approaches for most analytical results. Keywords: starch granule; in vitro digestion; experimental design; data analysis; kinetic model; starch digestion; amylase; in vitro assay 1. Introduction The study of starch hydrolysis by amylase, or amylolysis, in human health is central for assessing the role of starch in foods [1]. Amylolysis is also important for many industrial processes such as malting and fermentation, as well as the production of glucose, glucose syrups and bioethanol [2]. Finally, it is a fundamental natural process by which the energy stored in granular starch is delivered for the plant metabolism. Starch comprises two polymers of glucose residues: amylose and amylopectin. Amy- lopectin represents by far the major fraction of native starch—75–90% of the relative dry weight of wild-type starches—while amylose is the minor fraction [3]. Both amylose and amylopectin are essentially composed of linear long chains of α-(1,4)-linked glucosyl units with α-(1,6)-branched points [4]. Amylose is essentially linear, with less than 1% α-(1,6)- branched points, while amylopectin is a highly-branched shorter α-(1,4) chain of glucosyl units with 5% α-(1,6)-branches [5]. Native starch appears in the form of granules with alternating amorphous and semi-crystalline growth rings extending from the core of the granule, which are called hilum. Starch granules exhibit a hierarchical structure in which Foods 2022, 11, 1223. https://doi.org/10.3390/foods11091223 https://www.mdpi.com/journal/foods